The Role of the Tyrosine-Based Sorting Signals of the ORF3a Protein of SARS-CoV-2 on Intracellular Trafficking, Autophagy, and Apoptosis

The open reading frame 3a (ORF3a) is an accessory transmembrane protein that is important to the pathogenicity of SARS-CoV-2. The cytoplasmic domain of ORF3a has three canonical tyrosine-based sorting signals (YxxΦ; where x is any amino acid and Φ is a hydrophobic amino acid with a bulky -R group). They have been implicated in the trafficking of membrane proteins to the cell plasma membrane and to intracellular organelles. Previous studies have indicated that mutation of the 160YSNV163 motif abrogated plasma membrane expression and inhibited ORF3a-induced apoptosis. However, two additional canonical tyrosine-based sorting motifs (211YYQL213, 233YNKI236) exist in the cytoplasmic domain of ORF3a that have not been assessed. We removed all three potential tyrosine-based motifs and systematically restored them to assess the importance of each motif or combination of motifs that restored efficient trafficking to the cell surface and lysosomes. Our results indicate that the YxxΦ motif at position 160 was insufficient for the trafficking of ORF3a to the cell surface. Our studies also showed that ORF3a proteins with an intact YxxΦ at position 211 or at 160 and 211 were most important. We found that ORF3a cell surface expression correlated with the co-localization of ORF3a with LAMP-1 near the cell surface. These results suggest that YxxΦ motifs within the cytoplasmic domain may act cooperatively in ORF3a transport to the plasma membrane and endocytosis to lysosomes. Further, our results indicate that certain tyrosine mutants failed to activate caspase 3 and did not correlate with autophagy functions associated with this protein.


INTRODUCTION
ORF3a mutants for LC3-II formation and levels of SQSTM1/p62. Compared with HEK293 291 cells transfected with the empty vector, transfection of cells with the vector expressing 292 unmodified ORF3a resulted in an increase in LC3-II but with little degradation of p62. 293 These results were comparable to previous studies (7, 21, 23). Analysis of ORF3a and 294 the ORF3a mutants for LC3-II lipidation indicated that ORF3a and the tyrosine mutants 295 had increased lipidation of the LC3-I to LC3-II when compared with the empty vector 296 control (Fig. 13). Examination of cells transfected with vectors expressing the unmodified 297 ORF3a or ORF3a with amino acids substitutions of the tyrosine residues revealed that 298 p62 was not degraded in cells but was increased over the empty vector control (Fig. 13). 299 300 301 302 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 15 DISCUSSION 303

304
Previous studies have shown that the ORF3a viroporin of SARS-CoV-2 is a 305 virulence factor in pathogenesis (4). Using the hACE-2/mouse model, deletion of ORF3a 306 from SARS-CoV-2 did not result in a significant reduction in virus titers in cell culture but 307 had a profound effect on in vivo lung pathology (4), indicating that the ORF3a contributes 308 to in vivo pathology. ORF3a has been reported to have several biological functions that 309 could impact the pathogenesis in the hACE-2/mouse model. These functions include an 310 ion channel activity, the ability to induce apoptosis, and to disrupt the autophagy pathway. 311 Thus, identification of those protein domains that are important to ORF3a trafficking and 312 biological functions in vivo could possibly aide in the development of more robust 313 vaccines that result in long-term immunity against this viral disease. 314 In a recent study, the SARS-CoV was shown to cause apoptosis through both the 315 intrinsic and extrinsic pathways while the SARS-CoV-2 ORF3a was shown to cause 316 apoptosis through an extrinsic pathway (6). This was based on the findings that the 317 presence of SARS-CoV-2 ORF3a resulted in the cleavage of pro-caspase 9 and pro-318 caspase 8 of the extrinsic pathway (6). These investigators showed that a tyrosine to 319 alanine substitution within the tyrosine-based sorting motif ( 160 YNSV 163 ) resulted in a 320 protein (ORF3a-YA) that no longer trafficked to the cell plasma membrane and did not 321 induce apoptosis, suggesting that cell surface expression was necessary for SARS-CoV-322 2 ORF3a induced apoptosis. However, these investigators also showed that ORF3a-YA 323 was not associated with membrane fractions of the cell. This is puzzling as this tyrosine 324 to alanine substitution should not affect the biosynthesis and translocation of this protein 325 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 across the RER membrane. Further, it is unclear why these investigators did not analyze 326 the other potential tyrosine-based sorting motifs within the cytoplasmic domain. 327 In this study, we have expanded on the studies of the tyrosine-based motifs of the 328 SARS-CoV-2 ORF3a protein. Analysis of the cytoplasmic domain (CD) of these ORF3a 329 proteins revealed that more than one potential tyrosine-based sorting motif exists in the 330 CD of these proteins (SARS-CoV-2: 160 YNSV 163 , 211 YYQL 214 , and 233 YKNI 236 , SARS-331 CoV: 160 YNSV 163 ,200 YVVV 203 ,and 211 YYQL 214 ). The central question we addressed is 332 whether one or more of these other potential tyrosine motifs contribute to the trafficking 333 of the SARS-CoV-2 ORF3a to the cell surface and lysosomes, and if so, do they also 334 contribute to the biological functions of this protein? We used a strategy in which we first 335 eliminated all three tyrosine-based sorting signals by altering each tyrosine to an alanine. 336 The resulting protein, ORF3a-ΔYxxΦ, was detectable in several organelles of the 337 secretory pathway (ER, ERGIC, cis-medial Golgi, trans Golgi and TGN). However, 338 ORF3a-ΔYxxΦ was neither detectable at the cell surface nor did it co-localize with LAMP-339 1, a marker for late endosomes/lysosomes. Conversely, the intact ORF3a was easily 340 detected at the cell plasma membrane. To determine the importance of each individual 341 tyrosine motif alone or the combinations of two motifs, we generated mutants with one or 342 two tyrosine-based sorting motifs intact. For analysis of intracellular trafficking, we used 343 either: a) plasmids expressing proteins tagged with fluorescent proteins that localize in 344 different organelles; or b) antibodies specific for organelle specific proteins. We reasoned 345 that if the 160 YNSV 163 motif was critical to transport to the surface, the mutant with just 346 160 YNSV 163 motif intact (ORF3a-Y160) should be transported to the cell surface. Our 347 results indicated that ORF3a-Y160 was inefficiently transported to the cell surface, 348 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 indicating that the 160 YNSV 163 motif by itself does not dictate efficient transport to the cell 349 surface. Our results indicated that the ORF3a with only the 211 YYQL 214 motif intact 350 (ORF3a-Y211) was efficiently transported to the cell surface. Finally, our data also 351 indicated that mutant ORF3a-Y160,211 was also transported to the cell surface 352 suggesting that these two motifs may act cooperatively to enhance the transport of 353 ORF3a to the cell surface or they do not interfere each other. Finally, the 233 YKNI 236 motif 354 did not appear to be involved in transport to the cell plasma membrane. This is based on 355 the results with ORF3a-Y160,233, ORF3a-211,233, and ORF3a-Y233, which were not 356 detected at the cell plasma membrane. Also, the tandem presence of Y211 and Y233 357 interfered with the transport of ORF3a-Y211,233 to the plasma membrane. This suggests 358 that the Y233 motif may interfere in the trafficking of ORF3a to the cell surface in the 359 presence of one other tyrosine motif (211) and that the presence of both Y160 and Y211 360 motifs is required to overcome the influence of Y233 motif. Alternatively, the Y233 motif 361 may be necessary for endocytosis, perhaps to the lysosomes of the cell, which has been 362 well documented (7, 21, 25). Thus, while the results of Ren and colleagues (6) were 363 correct with their Y160A mutant (the equivalent to our ORF3a-Y211,233 mutant, it was 364 likely not due to the disruption of the 160 YNSV 163 motif. One caveat to our studies is that 365 the substitution of the tyrosine residues with alanines may have changed the overall 366 structure of the CD. However, comparable results were obtained when we substituted the 367 tyrosine residues with structurally similar phenylalanines, suggesting that an overall 368 change in the CD was not likely the cause for our results (Fig. 10). While our studies 369 concentrated on the ORF3a of SARS-CoV-2, it is of interest that of the three tyrosine 370 motifs examined in 14 SARS-CoV-2 and SARS-CoV-2-like isolates, the 160 YNSV 163 motif 371 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 was 100% conserved while the 211 YYQL 214 motif was conserved in 13 of 14 isolates 372 Autophagy is a conserved cellular process of intracellular degradation of 374 senescent or malfunctioning organelles to maintain intracellular homeostasis (26)(27)(28)(29). 375 Autophagy occurs in response to different forms of stress, including nutrient deprivation, 376 growth factor depletion, infection, and hypoxia. This process can target viral components 377 or even full viruses for lysosomal degradation (30). Most successful viruses developed 378 strategies to avoid degradation by autophagy or have evolved to exploit components of 379 the autophagic machinery to enhance their replication and to mediate membrane 380 trafficking and fusion processes. In a recent study, the role of different SARS-CoV-2 381 proteins revealed that E, M, ORF3a, ORF7a and Nsp15 affected autophagy (31,32). 382 These investigators found that the number of LC3-II positive autophagosomes was 383 decreased in the presence of Nsp15 while expression of E, M, ORF3a, and ORF7a 384 caused a strong accumulation of membrane-associated LC3-II (31). ORF3a increases 385 the conversion of microtubule-associated protein light chain 3 (LC3B-I) to the lipidated 386 form, LC3B-II, while the level of SQSTM1/p62 did not decrease, indicating that ORF3a 387 blocks autophagosome-lysosome fusion. Relating to this incomplete autophagy, late 388 endosomal ORF3a interacts directly with and sequestrates VPS39 of the homotypic 389 fusion and protein sorting (HOPS) complex (7, 21). This prevents the HOPS complex from 390 interacting with the autophagosomal SNARE protein STX17 and blocks the assembly of 391 the STX17-SNAP29-VAMP8 SNARE complex, which mediates autophagosome/ 392 amphisome fusion with lysosomes (22, 33). Finally, one study has suggested that ORF3a 393 may mediate virus release from lysosomes. However, the extent to which this occurs 394 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. ; within infected cells is still unknown (34). We analyzed LC3-II lipidation of the unmodified 395 ORF3a and our mutants. Our results indicated that lipidation of LC3-I was similar in 396 ORF3a-transfected and ORF3a mutant transfected cells, indicating that the tyrosine 397 motifs did not play a crucial role in this process. Upon fusion of LC3-II decorated 398 autophagosomes with lysosomes, the autophagic receptor p62 is degraded (autophagic 399 turnover). Analysis of p62 levels in cells expressing the unmodified ORF3a or the tyrosine 400 motif mutants was essentially the same with the unmodified ORF3a and the tyrosine motif 401 mutants, regardless of if they were transported to the cell surface or retained in 402 intracellular compartments. 403 In addition to its role in transport to the cell plasma membrane, the same sorting 404 signals are also involved in targeting membrane proteins to the lysosomes (13, 35, 36). 405 Previous studies have shown that ORF3a interacts with the components of the lysosome 406 to prevent fusion of lysosomes with autophagosomes (7, 21, 25). Lysosomal membrane 407 proteins are targeted to lysosomes using either direct or indirect pathways. With the direct 408 pathway, lysosomal membrane proteins are transported from the TGN to either early or 409 late endosomes and then to lysosomes. In the indirect pathway, lysosomal proteins are 410 first transported from the TGN to the plasma membrane followed by endocytosis to early 411 endosomes and eventual delivery to late endosomes/lysosomes. Lysosomal membrane 412 proteins possess sorting signals in their cytoplasmic domains that mediate both lysosomal 413 targeting and rapid endocytosis from the cell surface. These signals have been best 414 characterized for members of the lysosomal-associated membrane proteins (LAMP) and 415 lysosomal integral membrane proteins (LIMP) but are also present in other lysosomal 416 membrane proteins. Like other TGN sorting and endocytic signals, the majority of 417 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. ; lysosomal targeting signals belong to either the YxxΦ or [DE]xxxL [LI] types but also have 418 other features that make them functional for lysosomal targeting. One of the most 419 important features is the placement of either type of signal close (often 6-13 residues) to 420 the transmembrane domain (37, 38). The use of multiple YxxΦ signals for targeting 421 proteins to the lysosomes has been reported. SID1 transmembrane family member 2 422 (SDIT2) is an integral membrane protein of lysosomes that mediates the translocation of 423 RNA and DNA across the lysosomal membrane during RNA and DNA autophagy (RDA), 424 a process in which RNA or DNA is directly imported into lysosomes and degraded (39, 425 40). Human and mouse SIDT2 homologs show 95% sequence identity across the entire 426 protein (832 amino acids) and 100% identity at the C-terminal 100 amino acids (41). With 427 SIDT2, localization to the lysosomal membrane was mediated by three cytosolic YxxΦ 428 motifs located between transmembrane 1 and 2, and SIDT2 interacts with AP-1 and AP-429 2 through the Y359GSF motif (42). Our data indicated that tyrosine-based sorting signals 430 (YxxΦ) at positions 160 and 211 were necessary for ORF3a trafficking to lysosomes, 431 which coincidentally were the same motifs required for transport to the cell plasma 432 membrane. This suggests that ORF3a is likely transported to the cell surface prior to 433 endocytosis and targeting lysosomes. This is of importance as it was recently reported 434 that β-coronaviruses such as SARS-CoV-2 use the endosomal pathway and lysosomes 435 for egress rather than the secretory pathway (34). In this study, the investigators showed 436 that ORF3a co-localized with lysosomes and presented evidence that ORF3a caused 437 lysosome de-acidification, presumably through its viroporin activity. This was determined 438 by the expression of ORF3a and staining transfected cells with Lysotracker Red 439 which is a cell permeable, acidophilic dye that accumulates in acidic organelles. Under 440 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. ; https://doi.org/10.1101/2023.07.24.550379 doi: bioRxiv preprint these conditions, the Lysotracker red fluorescence was diminished in the presence of 441 ORF3a, suggesting that ORF3a may have caused the de-acidification of the lysosomes 442 via a proton channel. Whether ORF3a is a proton ion channel remains to be determined. 443 Recently, SARS-CoV-2 ORF3a was expressed in Spodoptera frugiperda, reconstituted 444 into liposomes, and single-channel currents were recorded from excised patches (4). 445 ORF3a behaved as a cation channel with a large single-channel conductance (375 pA) 446 that had a modest selectivity for Ca +2 and K + over Na + ions and was not blocked by Ba ++ , 447 which was the case for the SARS-CoV channel (26). However, no studies have reported 448 SARS-CoV-2 ORF3a being a proton ion channel. This differed from the SARS-CoV 449 ORF3a, which could induce apoptosis via the intrinsic pathway. The apoptosis function 450 of SARS-CoV ORF3a has been reported to involve the ion channel activity of this protein 451 (43). 452 In addition to potential effects of the ORF3a tyrosine motifs on the intracellular 453 transport to the cell surface and lysosomes, the SARS-CoV ORF3a induces NF-kB 454 activation, chemokine production, Golgi fragmentation, accumulation of intracellular 455 vesicles, and cell death (44, 45). Both SARS-CoV and SARS-CoV-2 ORF3a proteins 456 have been implicated in the induction of apoptosis (6, 44, 46, 47). In the most recent 457 study, investigators found a correlation between SARS-CoV-2 ORF3a-induced apoptosis 458 and cell plasma membrane expression (6). Mutation of the tyrosine of 160 YNSV 163 resulted 459 in neither plasma membrane expression nor apoptosis (6). However, the use of 460 established cellular markers to determine the intracellular site of expression was not 461 performed. We analyzed the level of apoptosis caused by the unmodified ORF3a and 462 various tyrosine mutants. We analyzed the levels of cleavage of procaspase 3 to caspase 463 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. ;

22
3, which is an effector caspase. We observed that ORF3a-Y160, which we found was 464 not observed at the cell surface, induced caspase 3 activity while mutant 465 which was expressed at the cell surface, did not induce caspase 3 activity, indicating 466 there was no correlation between cell surface expression and apoptosis. This was 467 reinforced with other ORF3a mutants (Fig. 12). was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. ; was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

MATERIALS AND METHODS
The copyright holder for this preprint (which this version posted July 24, 2023. ; https://doi.org/10.1101/2023.07.24.550379 doi: bioRxiv preprint 24 Site-directed mutagenesis of ORF3a. For site-directed mutagenesis, the pcDNA3.1(+) 502 vector containing the SARS-CoV-2 HA-ORF3a gene was used in site-directed 503 mutagenesis using a QuikChange II site-directed mutagenesis kit (Agilent) according to 504 the manufacturer's protocol. A similar mutant was constructed using ORF3a with C-505 terminal HA tag. We found no differences in the intracellular localization of the ORF3a 506 with C-or N-terminal HA tags (data not shown). For construction of the ORF3a-ΔYxxΦ, 507 the tyrosine residues of the three potential tyrosine signals ( 160 YNSV 163 , 211 YYQL 214 , and 508 233 YNKI 236 ) were changed to alanine residues ( 160 ANSV 163 , 211 AYQL 214 , 233 ANKI 236 ). The 509 ORF3a-ΔYxxΦ gene was sequenced to ensure that the desired changes were made and 510 that no unwanted changes occurred during the mutagenesis process. Using ORF3a-511 ΔYxxΦ, individual alanine residues were changed back to tyrosine residues to yield 512 ORF3a-Y160, ORF3a-Y211, and ORF3a-Y233. ORF3a proteins with combinations of two 513 tyrosine motifs were generated from the above single mutants to yield ORF3a-Y160,211, 514 ORF3a-Y160,233, and ORF3a-Y211,233. Again, all were sequenced to ensure that the 515 desired changes were made and that no unwanted changes occurred during the 516 mutagenesis process. 517 518 Immunofluorescence studies. To examine the intracellular localization of the SARS-519 CoV-2 ORF3a proteins, COS-7 cells grown on 13 mm coverslips were transfected with 520 either the empty pcDNA3.1(+) vector, pcDNA3.1(+) expressing the unmodified ORF3a-521 HA, or the same vector expressing the various SARS-CoV-2 ORF3a mutants. Vectors 522 were transfected into COS-7 cells using the Turbofect transfection reagent 523 (ThermoFisher) according to the manufacturer's instructions. With other experiments, 524 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. ; https://doi.org/10.1101/2023.07.24.550379 doi: bioRxiv preprint vectors expressing unmodified ORF3a-HA or ORF3a mutants were co-transfected with 525 vectors expressing various fluorescent marker proteins as described above. At 48 h post-526 transfection, cells were washed three times in PBS, fixed in 4% paraformaldehyde 527 (prepared in PBS) for 15 minutes, permeabilized with 0.1% Triton X-100 in PBS, and 528 blocked for one hour with 22.5 mg/mL glycine and 0.1% BSA in PBST. The cultures were 529 then incubated at 4C overnight with a mouse monoclonal antibody against HA-tag 530 (Thermo-Fisher, antibody 2-2.2.14, #26183) and one of the following rabbit polyclonal or 531 monoclonal antibodies: a) ERGIC53; b) Golgin-97 (trans Golgi marker; Abcam, ab84340) 532 or c) LAMP-1 (late endosome/lysosome marker; CST, D2D11). The cells were washed in 533 PBS and incubated with a secondary goat anti-rabbit antibody conjugated to 534 AlexaFluor™-488 (Invitrogen, A11008) and a chicken anti-mouse conjugated to 535 AlexaFluor™-594 (Invitrogen, A21201) for 1 h. Cells were counterstained with DAPI, and 536 the coverslips were mounted on glass slides with ProLong TM Diamond Antifade Mountant 537 (ThermoFisher, P36961). The coverslips were viewed with a Leica TCS SP8 Confocal 538 Microscope with a 100X objective and a 2X digital zoom using the Leica Application Suite 539 X (LASX). A 405nm filter was used to visualize DAPI staining, a 488nm filter was used to 540 visualize the organelle markers (to ER, ERGIC, cis/medial Golgi, trans Golgi, TGN, and 541 late endosomes/lysosomes) and a 594nm filter was used to visualize the ORF3a-HA 542 protein. To examine co-localization ORF3a proteins with mitochondria, COS-7 cells were 543 co-transfected with vectors the ORF3a proteins and the vector or 4xmts-Neon-Green 544 (mitochondria; Addgene, #98876). At 48 h post-transfection, cells were fixed, 545 permeabilized and stained for ORF3a-HA proteins as described above. A minimum of 50 546 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. Invitrogen) in PBS containing 1% BSA 1h at room temperature. Cells were permeabilized 559 with PBS containing 0.2% Triton X-100 for 15 min and blocked with PBS containing 22.5 560 mg/mL Glycine, 1% BSA and 0.1% Tween-20. Cells were incubated with a 1:400 dilution 561 of the primary antibody (mouse anti-HA, 2-2.2.14) in PBS containing 1% BSA and 0.1% 562 Tween-20 for 1h at room temperature, washed three times and incubated with second 563 secondary antibody (Rabbit anti-mouse-AF488, A11059, Invitrogen) in PBS containing 564 1% BSA 1h at room temperature. Finally, cells were washed three times, stained with 565 DAPI 5 min and mounted using Prolong Diamond Antifade Mountant (Invitrogen, 566 P36961). Cells were examined as described above. 567 568 Apoptosis assays. We analyzed the ability of ORF3a-HA and the mutants for the ability 569 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. ; https://doi.org/10. 1101/2023 to induce apoptosis in cells. We used a colorimetric caspase-3 assay protocol (EnzChek 570 Caspase-3 Assay Kit #1, with the Z-DEVD-AMC substrate) based on the formation of the 571 chromophore 7-amido-4-methyl coumarin (AMC) by cleavage from the labeled substrate 572 Z-DEVD-AMC. HEK293 cells were transfected with either the empty vector, one 573 expressing the unmodified ORF3a-HA or the ORF3a mutants. At 48 h, cells were lysed 574 and assayed for caspase 3 activity according to the manufacturer's instructions. The 575 fluorescence of the AMC was quantified using a microtiter plate reader at 340 nm 576 excitation and 441 nm emission. 577 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023.  was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023.  was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. ; https://doi.org/10.1101/2023.07.24.550379 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. ; https://doi.org/10.1101/2023.07.24.550379 doi: bioRxiv preprint signals that were unchanged were bolded and underlined. Those potential tyrosine-based 766 sorting signals that were altered are boxed and bolded with the amino acid changes 767 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. immunostained with an antibody against the HA-tag followed by a secondary antibody 882 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. Cells were washed, mounted, and examined using a Leica TSP8 laser scanning confocal 904 microscope. Shown are individual red and green images and merged images of the red, 905 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. were fixed, permeabilized, and blocked. Cells were reacted with a mouse monoclonal 927 antibody against the HA-tag and a rabbit antibody against LAMP-1 overnight, washed, 928 . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. ; https://doi.org/10. 1101/2023 and reacted with an appropriate secondary antibody tagged with Alexa Fluor 594 (for HA) 929 and Alexa Fluor 488 (for LAMP-1) for 1 h. Cells were washed and counter-stained with 930 DAPI (1 μg/ml) for 5 min. Cells were viewed using a Leica TC8 confocal microscope and 931 at least 50 cells were examined for co-localization with the LAMP-1 marker. Panel A. HA-932 was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023 subjected to SDS-PAGE and proteins transferred to PVDF membranes as described in was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023  B. Figure 2. The ORF3a mutants analyzed in this study. Panel A. The unmodified ORF3a and the mutants were constructed for this study. The potential tyrosine-based sorting signals that were unchanged were bolded and underlined. Those potential tyrosine-based sorting signals that were altered are boxed and bolded with the amino acid changes underlined. Panel B.
Expression of the ORF3a and its mutants. HEK293 cells were transfected with vectors expressing the unmodified ORF3a and the seven mutants were analyzed. Proteins were separated by SDS-PAGE, transferred to membranes, and analyzed in immunoblots using an antibody directed against the C-terminal HA-tag. β-actin served as a control for loading of samples.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023  . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

RER
The copyright holder for this preprint (which this version posted July 24, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023  . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

RER
The copyright holder for this preprint (which this version posted July 24, 2023. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  Figure 9. Surface immunostaining of cells transfected with vectors expressing ORF3a mutants with two tyrosine motifs intact. COS-7 cells were transfected with vectors expressing each of the ORF3a mutants. At 24 h post-transfection, cells were immunostained with an antibody against the HA-tag followed by a secondary antibody tagged with Alexa Fluor 594. The cells were washed three times and permeabilized as described in the Materials and Methods section. Permeabilized cells were then reacted with the same primary antibody and a secondary antibody tagged with Alexa Fluor 488. Cells were washed, mounted, and examined using a Leica TSP8 laser scanning confocal microscope. Shown are individual red and green images and merged images of the red, green, and blue channels. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  Figure 11. Co-localization of ORF3a and the tyrosine-based sorting signal mutants with LAMP-1. COS-7 cells were transfected with the empty vector or the same vector expressing ORF3a or individual ORF3a mutants. At 48 hr. post-transfection, the cells were fixed, permeabilized, and blocked. Cells were reacted with a mouse monoclonal antibody against the HA-tag and a rabbit antibody against LAMP-1 overnight, washed, and reacted with an appropriate secondary antibody tagged with Alexa Fluor 594 (for HA) and Alexa Fluor 488 (for LAMP-1) for 1 h. Cells were washed and counter-stained with DAPI (1 μg/ml) for 5 min. Cells were viewed using a Leica TC8 confocal microscope and at least 50 cells were examined for co-localization with the LAMP-1 marker. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. Controls included transfected cells treated with 2 μM staurosporine (STS) for 18h, and transfected cells treated with the pan-caspase inhibitor Z-VAD-FMK (InvivoGen). The assays were performed a minimum of three times and analyzed for statistical significance using Students' t-test.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. ; https://doi.org/10.1101/2023.07.24.550379 doi: bioRxiv preprint . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. . CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 24, 2023. ; https://doi.org/10. 1101/2023